Day 1 Lecture Notes

Steve Williams, Smith College

June 6, 2004

Recommended text: Genes by Benjamin Lewin. Recommended laboratory manual: Current Protocols in Molecular Biology by F.M. Ausubel et al. (the "Red Book").

This session is the 51st of the New England Biolabs Summer School, which is in its 19th year. There are about 2500 graduates.

Expt. 1: The reverse transcriptase (RevT) family makes up 17% of the human genome and is the largest gene family. Two new papers about the role of RevT appeared in Nature during the first week of June. The high copy number of RevT makes experiments with it easier. The technique used for lower-copy genes is the same.

The bacteriophage lambda infects E. coli. We will clone our RevT gene into lambda and then subclone into a plasmid.

Expts. 2 and 3: We will study the Ttr (transthyretin) gene, which was previously called both Tth and prealbumin. The name prealbumin comes from running just above the common protein albumin on a gel.

Expt. 4: A cDNA (copy DNA) library contains about 1% of the the DNA that a genomic library would have since it comes only from expressed genes. The direct source of a cDNA library is mRNA (messenger RNA).

Expt. 5: We will all measure our own "DNA fingerprints." The chances of two individuals having identical DNA fingerprints is 1 in 82x10⁹.

To begin Expt. 1, we will cut mouse genomic DNA (DNA from a chromosome, not from mRNA) with the restriction enzyme EcoR1. EcoR1 cuts only the sequence GAATTC between the G and the first A. While not 100% efficient ("No biological reaction runs to 100% completion"), EcoR1 should in principle cut the DNA everywhere this sequence occurs. The starting DNA in Expt. 1 is typically 1x10⁵ base-pairs (bp) long and after cutting will be more like 3000 bp in length. These smaller pieces are more suitable for genomic library analysis.